The emerging threat of bioterrorism has created a need for rapid diagnostic assays of pathogenic bacteria. To be maximally effective, assays must not only be rapid, sensitive, and specific, but also sufficiently simple and robust to be used in local healthcare settings, as well as in regional laboratories. Assays must detect all bacteria that are classified as select agents, and should also have the capacity to identify common infectious diseases. This latter feature will encourage both familiarity with the assays and their widespread distribution, assuring their availability if a bioterrorist attack occurs. We propose the development of novel approaches to real-time PCR that vastly expand the number of different pathogens that can be detected in a single assay well. These assays will have the same sensitivity and robustness as the current generation of real-time polymerase chain reaction (PCR) methods, but will have a dramatically expanded ability to distinguish different pathogen-specific nucleic acid sequences. Our laboratories have pioneered molecular beacons as sequence-specific hybridization probes for use in real-time PCR assays. Here, we propose the development of a new paradigm for the use of molecular beacons that will enable highly multiplexed detection. We will replace specific molecular beacons with mixtures of molecular beacons that act in concert. Sequences will be identified by detecting "fluorescence signatures" generated by multiple probes that hybridize to DNA sequences in characteristic fashions. The switch from single-probe identification to multi-probe, pattern-based identification will enable us to develop highly multiplexed assays using only a small number of probes or colors. We propose two related approaches. We will create a "sloppy" molecular beacon assay that will use six differently colored molecular beacons to distinguish among 60 or more different target sequences. We will also develop a "color-triplet coding" format that will enable unique labeling of as many as 56 different molecular beacons in the same assay well, utilizing only eight differently colored fluorophores. A distinguishing feature of these approaches is that they are based on real-time PCR technology, which has already been reduced to practice in commercial assays. Our specific aims: 1. To develop sets of "universal" PCR primers that amplify species-specific DNA sequences from all select bacterial agents and common bacterial pathogens. 2. To develop PCR assays that are able to distinguish over 60 different species-specific DNA sequences in a single assay well, utilizing only five differently colored, semi-specific "sloppy" molecular beacons. 3. To develop PCR assays that utilize "color-triplet coding" to uniquely label as many as 56 different species-specific molecular beacons.